Modulation of NF-κB signaling pathway in murine lymphoma cells. (A) Immunoblot analysis of TD-IκBα expression in transduced murine lymphoma cell line. Cells were spin-infected with retroviral vectors IEGZ-empty or IEGZ-TD-IκBα and protein extracts were isolated 24 hours after infection. (B) EMSA of NF-κB binding activity in transduced lymphoma cell lines. NF-κB activation after doxycycline-mediated MYC inactivation was detectable only in IEGZ-empty– but not in IEGZ-TD-IκBα–transduced cells. Doxycycline (2 μg/mL) was added for 12 or 24 hours, respectively. (C) Immunoblot analysis of CA-IKK2 expression in transduced lymphoma lines. Protein extracts of transduced cells were isolated 24 hours after the infection. EMSA of the extracts displays induction of NF-κB binding in CA-IKK2–expressing cells. (D) Morphologic examination of transduced cell line 5522. Bright field and fluorescence microscopy of transduced cells 2 days after infection. Images were taken with a Leica DMIRBE microscope 10× NA = 0.3 PH1 acquired with a Hamamatsu digital camera C4742-95 (Hamamatsu Photonics) and Open Lab software Version 4.0.4 (Improvision). (E) Flow cytometric determination of changes in the GFP-positive fractions in transduced cultures. Murine lymphoma cells were transduced with IEGZ-empty (◇), IEGZ-TD-IκBα (□), or IEGZ-CA-IKK2 (▲), and percentage of GFP⁺ cells was measured by flow cytometer at the indicated time points after retroviral infection. Percentage of GFP⁺ cells at day 1 after infection was set to 100% to facilitate comparisons. (F) Apoptosis detection of murine lymphoma cell line transduced with IEGZ-empty, IEGZ-TD-IκBα, or IEGZ-CA-IKK2. At day 3 after the infection the cells were stained and analyzed for annexin-V–APC and 7-AAD binding. (G) In vivo tumor competition assay. Murine B-lymphoma cell line 5522 was transduced with either IEGZ-empty or TD-IκBα. Transduced GFP⁺ cells were then mixed with parental nontransduced cells at a ratio of 1:10. Cells of mixed populations (10⁷) were injected intraperitoneally into syngeneic recipient mice and lymphocytes were isolated from ascites 1 week later. Flow cytometric determination of the GFP⁺ fractions is shown for the cells before transplantation (top panels) and after isolation of ascites of recipient mice (bottom panels). (H) Statistical analyses of competitive tumor transplantation. Mean values of the percentage of the GFP⁺ fraction of cells isolated from recipient mice (n = 6 for each group) detected by flow cytometry.